There are a number of biological and medical procedures that are currently impractical due to limitations in cell and particle analysis technology. Examples of such procedures include battlefield detection and monitoring of both known and unknown toxins, non-invasive prenatal genetic testing and routine cancer screening via the detection and analysis of rare cells (i.e., cells having a low rate of occurrence) in peripheral blood, and drug discovery via high throughput cell assays.
New medical and biological procedures increasingly require more advanced cell analysis capabilities than currently exist. One example is the analysis of changes in the genetic constitution of tumor cells for the optimization of chemotherapy. Tumor cells may exhibit unusual DNA changes, such as a variation in the number of chromosomes, the amplification of chemotherapy-resistance genes, or changes in the regulation of gene expression. These changes can be detected using Fluorescence In-Situ Hybridization (FISH) probes that bind to specific DNA sequences within cells. FISH analysis requires an accurate determination of the number of distinct FISH locations within the nucleus of a cell, ideally in three dimensions. Commonly assigned U.S. patent application Ser. No. 09/490,478 describes the use of a stereoscopic imaging apparatus to view fluid-suspended cells from multiple angles, with a high numerical aperture for the accurate enumeration of FISH spots within a cell. This technique can be applied to a slide-mounted sample or a sample on a micro-fluidic chip, but generally with lower numerical apertures, due to the difficulty of coupling orthogonal collection systems to the flat sample substrate. Clearly, it would be preferable to perform three-dimensional (3D) imaging of cells on slides or in micro-capillaries with a single collection system in order to enable light collection with a high numerical aperture.
The most accurate determinations of FISH spot counts in cells on flat substrates are currently based on high-resolution fluorescence images taken at different focal planes across the depth of the cell. The resulting set of two-dimensional (2D) images are reconstructed into a three-dimensional (3D) representation of the cell, and FISH spots are counted both within and across the image planes to ensure that superimposed FISH spots within a single image are resolved across the multiple images. While such image stacking techniques can effectively resolve superimposed FISH spots, existing systems for 3D cell imaging are slow, often requiring several minutes to create each 3D composite. As a result, the various 2D images are gathered at widely different times, and changes in the cell over the course of the imaging process alter the resulting 3D representation. Further, such systems cannot tolerate movement of the cells during the imaging process, which limits their application to fixed cells that are immobilized on slides. An improved system would allow the rapid 3D imaging of cells, including cells in motion.
Accordingly, it will be apparent that an improved technique is desired that resolves the limitations in analyzing the three-dimensional features of both stationary and moving cells imposed by the conventional approaches discussed above. In addition, a new approach developed to address these problems in the prior art should also have application to the analysis of other types of objects besides cells and should be amenable to implementation in different configurations to meet the specific requirements of disparate applications of this technology.